The present invention is a family of eukaryotic expression plasmids, and immunization strategies, useful for genetic immunization. Such molecules and methods for use are useful in biotechnology, gene therapy, cancer and agriculture.
With the invention in mind, a search of the prior art was conducted. DNA vaccines (genetic vaccines) are a potential disruptive technology, that offer the promise of a new way to immunize humans (or animals) with materials that are entirely gene-based and expressed by the organism's own cells, making an ideal mimic of intracellular antigens.
Methods to improve immune responses to DNA vaccine plasmids are described in the art. For example, the efficacy of a DNA vaccine can be further improved, or tailored for systemic or mucosal immunity, or cancer, allergy, bacterial, intracellular parasite or viral targets, by; communization with costimulatory plasmids (e.g. IL12) to modulate the type of response (TH1 versus TH2 bias); cell death inhibitors or enhancers; or optimization of delivery (e.g. electroporation versus gene gun). Some such methods and molecules are described in Lemieux, P. 2002 Expert Rev. Vaccines 1: 85-93, Toka F N, Pack C D, Rouse B T. 2004 Immunological reviews 199: 100-112, and Gurunathan S, Klinman D M, Seder R A. 2000 Annu. Rev. Immunol. 18: 927-974 and are included herein by reference. DNA vaccination could also involve utilizing different delivery systems in the prime and the boost, as taught by Buchan S, Gronevik E. Mathiesen I, King C, Stevenson F K, Rice J. 2005 Immunol. 174: 6292-6298 or different injection sites, as taught by Pavlakis G N, Gragerov A, Felber B K. 2004 US Patent Application 2004/0241140.
DNA vaccination would significantly enhance the rapid deployment utility of DNA vaccines since development times for DNA vaccines are significantly shorter than those for protein or viral vector systems.
Current Obstacles
Protective immunity in humans and other primates has not been broadly obtained using DNA only vaccination. Primate efficacy has been obtained utilizing DNA vaccines in combination with a heterologous protein, inactivated organism, or viral vector boosting. Enhanced immune responses have also been reported when plasmid DNA and purified protein (corresponding to the protein encoded in the plasmid) (Dalemans W., Van Mechelen M V, Bruck C, Friede M. 2003 U.S. Pat. No. 6,500,432; Carrera S D, Grillo J M, de Leon LALP, Lasa A M, Feyt R P, Rodriguez A V, Obregon J C A, Rivero N A Donato GM 200420040234543; and Imoto J, Konishi E. 2005 Viral Immunol. 18: 205-212) or inactivated virus (Rangarajan P N, Srinivasan V A, Biswas L, Reddy G S. 2004 US Patent Application 2004/0096462) are mixed and coinjected.
However, using plasmids in combination with inactivated organisms, proteins or viral vectors in a vaccine (either as a mixture, or sequentially in a prime boost) eliminates most of the benefits of DNA vaccination, including improved safety, reduced cost, and rapid deployment.
DNA vaccines may be incrementally improved by the following methodologies:
Antigen expression: The art teaches that one of the limitations of DNA vaccination is that antigen expression is generally very low. Vector modifications that improve antigen expression (e.g. codon optimization of the gene, inclusion of an intron, use of the strong constitutive CMV or CAGG promoters versus weaker or cell line specific promoter) are highly correlative with improved immune responses (reviewed in Manoj S, Babiuk L A, Drunen S V, en Hurk L V. 2004 Critical Rev Clin Lab Sci 41: 1-39). A hybrid CMV promoter (CMV/R) with 5- to 10-fold improved expression improved cellular immune responses to HIV DNA vaccines in mice and nonhuman primates (Barouch D H, Yang Z Y, Kong W P, Korioth-Schmitz B, Sumida S M, Truitt D M, Kishko M G, Arthur J C, Miura A, Mascola J R, Letvin N L, Nabel G J. 2005 J Virol. 79: 8828-8834). A plasmid containing the woodchuck hepatitis virus posttranscriptional regulatory element (a 600 bp element that increases stability and extranuclear transport of resulting in enhanced levels of mRNA for translation) enhanced antigen expression and protective immunity to HA in mice (Garg S, Oran A E, Hon H, Jacob J. 2002 J Immunol. 173: 550-558). These studies teach that improvement in expression beyond that of current CMV based vectors may generally improve immunogenicity in humans.
The art teaches that plasmid entry into the nucleus is a limiting factor in obtaining antigen expression. Increasing nuclear localization of a plasmid through inclusion of NFκB binding sites or a SV40 enhancer improves antigen expression in vitro and in vivo; this is presumed due to binding of NFκB which then piggybacks the plasmid to the nucleus (Dean D A, Dean B S, Muller S, Smith L C. 1999 Experimental Cell Research 253: 713-722). However, NFκB is generally cytoplasmically localized, and transfer to the nucleus is both limited, tissue-specific, and dependent on stimulatory signals. This limits the utility of NFκB nuclear targeting to improve DNA vaccination.
TH1 or TH2 bias: The art teaches that shifting immune response to DNA vaccine expressed viral or other antigens from TH2 to TH1 is desirable, to elevate humoral and cellular responses; for other applications, such as allergy, a TH2 biased response is considered optimal. For example, CpG sequences (which promote TH1 response) improved antibody and CTL responses to influenza hemaglutinin (HA), and CTL responses to influenza nucleoprotein DNA vaccines injected IM (Lee and Sung, 1998). Communization with IL12 or IL15 TH1 adjuvants improves T cell responses to HA (Chattergoon M A, Saulino V, Shames J P, Stein J, Montaner L J, Weiner D B. 2004 Vaccine 22: 1744-1750; Kutzler M A, Robinson T M, Chattergoon M A, Choo D K, Choo A Y, Choe P Y, Ramamathan M P, Parkinson R, Kudchodkar S, Tamura Y, Sidhu M, Roopchand V, Kim J J, Pavlakis G N, Felber B K, Waldmann T A, Boyer J D, Weiner D B. 2005 J Immunol 175: 112-123) and antibody mediated protection (Operschall E, Pavlovic J, Nawrath M, Molling K. 2000 Intervirol 43: 322-330).
Immunostimulatory Adjuvants:
A number of microbial specific motifs have been identified that activate innate immunity through Toll like receptor (TLR) binding, for example, Tri-acyl lipopeptides (TLR-1/TLR2) peptidoglycan (TLR-2), dsRNA (TLR3), bacterial HSP60 or Lipopolysaccharide (LPS; TLR-4), flagellin (TLR5), Di-acyl lipopeptide (TLR-6) ssRNA (TLR-7, TLR-8) unmethylated CpG DNA (TLR-9). U-rich or U/G rich ssRNA TLR7/8 agonists have been identified that induce interferon responses (Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, Lipford G, Wagner H, Bauer S. 2004 Science 303: 1526-1529; Diebold S S, Kaisho T, Hemmi H, Akira S, e Sousa C R. 2004 Science 303: 1529-1531; Barchet W, Krug A, Cella M, Newby C, Fischer J A A, Dzionek A, Pekosz A, Colonna M. 2005 Eur. J. Immunol. 35: 236-242) as well as a sequence specific siRNA that induces interferon production from human and mice plasmacytoid dendritic cells through TLR-7 (Hornung V, Guenthner-Biller M, Bourquin C, Ablasser A, Schlee M, Uematsu S, Noronha A, Manoharan M, Akira S, de Fougerolles A, Endres S, Hartmann G. 2005 Nat. Med. 11: 263-270). A novel class of immunostimulatory nucleic acid, single stranded CpG RNA which does not require TLR-3, 7, 8 or 9 has also been identified (Sugiyama T, Gursel M, Takeshita F, Coban C, Conover J, Kaisho T, Akira S, Klimnan D M, Ishii K J. 2005 J Immunol. 174: 2273-2279).
These molecules can be utilized as adjuvants to improve DNA vaccination. However, exogenously applied adjuvant adds expense, complicates regulatory approval (an additional investigational entity in the vaccine) and requires high dosages since the adjuvant is not targeted (i.e. affects multiple cells in addition to cells containing the DNA vaccine); the high dose of untargeted adjuvant also presents special safety concerns (e.g. autoimmunity, sepsis)
Unmethylated CpG is present in the vector backbone of microbial produced plasmids and augmentation (CpG enriched plasmids) can be used to stimulate TH1 responsive innate immune signals through TLR-9. Unfortunately, these effects are observed only with high dosages, and CpG effects are minimal with advanced delivery methods which use economically low amounts of antigen (e.g. gene gun) as reflected by a TH2 biased response. As well, the overall poor immunological response to DNA vaccines in humans has been attributed, in part, to significantly reduced expression of TLR-9 in humans compared to mice.
Vector encoded protein TLR agonists potentially would induce the innate immune system at low dose, since the signal from these elements is “amplified” from the vector (rather than a fixed vector component such as CpG). Incorporation of a flagellin producing gene into the vector backbone activates innate immune responses and potentiated TH1 bias and cellular immune response to an antigen delivered by Gene Gun. This demonstrates the potential for utilization of amplifiable TLR agonists to potentiate low dose DNA vaccination (Applequist S E, Rollman E, Wareing M D, Liden M, Rozell B, Hinkula J, Ljunggren H G. 2005 J. Immunol. 175: 3882-3891). However, for inclusion of an innate immunity inducer in a DNA vaccine vector backbone there should be no associated adaptive immune response since this would limit repeat usage and generate variable results in a population due to attenuated responses in individuals with prior exposure (preexisting immunity). Vectors such as alphaviral replicons (which produce dsRNA adjuvant) or the flagellin producing vector described above contain one or more proteins that can induce adaptive immunity to vector components and are unsuitable for repeat application.
In summary, the art does not teach how to obtain immunostimulatory effects of amplified TLR agonists without the requirement for heterologous proteins in the vector backbone, which leads to adaptive immune responses.
Cell death: The art teaches that cell death can augment immune responses to antigens. IM injection of influenza HA and NP DNA vaccines codelivered with mutant caspases that promote slow cell death enhanced T cell responses and cellular immunity (Sasaki S, Amara R R, Oran A E, Smith J M, Robinson H L. 2001 Nat Biotechnol 19: 543-547). The immune response to HA and NP is also dramatically enhanced (compared to DNA vaccines) utilizing Semliki forest alphavirus replicon (suicide) vaccines (Berglund P, Smerdou C, Fleeton M N, Tubulekas I, Liljestrom P. 1998 Nature Biotech 16: 562-565) that induce apoptosis; cell death is critical for the improved immune response. Replicon vectors contain multiple viral replication proteins; immune response against these proteins may limit repeat usage. Apoptotic cell death has also been accomplished by coadministering Fas or mutated caspases 2 or 3, which enhances CTL responses to IM administered DNA vaccines. Coadministered caspases also improves immune responses to influenza HA DNA vaccine by Gene Gun (Sasaki et al, Supra 2001). The optimal condition may be to selectively kill muscle or keratinocyte cells (but not immune cells) for a source of antigen for dendritic or langerhans cells (Reviewed in Leitner W W, Restifo N P. 2003 J Clin invest 112: 22-24). This is not possible utilizing constitutive cell death promoting agents. Inhibition of apoptosis can also improve immune responses, wherein coadministering antiapoptotic Bcl-XL strongly enhanced T cell response after Gene Gun administration. This may reflect a benefit of prolonging dendritic cell lifespan. However, the use of cell death inhibitors may predispose cells to transformation (in the case of integrated plasmids) and increase cancer risk.
Cytoplasmic dsRNA activates PKR and RIG-1, which induces interferon production, inhibits protein synthesis thus reducing antigen production eventually leading to apoptotic cell death (reviewed in Wang Q, Carmichael G G. 2004 Microb. Molec. Biol. Rev. 68: 432-452). Cell death releases the dsRNA, which can then be taken up by cells, and further induce innate immune response by binding and stimulating endosomally localized TLR-3 (Reviewed in Schroder M, Bowie AG. 2005 Trends Immunol. 26: 462-468). The art teaches that this type of dsRNA stimulation occurs with alphavirus replicon vaccines. Alphavirus replicon (suicide) vaccines induce enhanced immune responses with 100-1000 fold less antigen compared to standard DNA vaccines (by IM injection). These vectors induce apoptosis, presumed through formation of dsRNA which activates antiviral pathways and eventually leads to apoptotic cell death (Leitner W W, Ying H, Driver D A, Dubensky T W, Restifo N P. 2000 Cancer Research 60: 51-55). Cell death is required for improved vaccine efficacy and is mediated by cytoplasmic replicon dsRNA; it is possible that dsRNA in apoptotic elements are phagocytosed by APC's, and induce innate immunity through the endosomal TLR-3 dsRNA recognition pathway. Codelivery of anti-apoptotic gene (Bcl-XL) reduced protection, despite increasing antigen production (Leitner W W, Hwang L N, Bergmann-Leitner E S, Finkelstein S E, Frank S, Restifo N P. 2004 Vaccine 22: 1537-1544; Leitner W W, Hwang L N, DeVeer M J, Zhou A, Silverman R H, Williams B R G, Dubensky T W, Ying H, Restifo N P. 2003 Nature Med 9: 33-39; Matsumoto S, Miyagishi M, Akashi H, Nagai R, Taira K. 2005 J Biol Chem 280: 25687-25696). However, a delivery dependent balance between cell death signals and optimal production of antigen is required, since suicide DNA vaccines are not effective with Gene Gun delivery (which targets dendritic cells) unless an anti-apoptosis gene is included (Kim T W, Hung C F, Juang J, He L, Hardwick J M, Wu T C. 2004 Gene Ther 11: 336-342).
Antigen targeting: Poor immunogenicity has partially been solved by altering the intracellular localization using targeting fusion tags. It is established in the art that fusion proteins that alter the intracellular localization of an antigen (e.g. from cytoplasmic to secreted) or otherwise target antigen presenting pathways alter the resulting immune response (reviewed in Gurunathan et al, Supra 2000). Molecules that can be used to alter intracellular trafficking or antigen presentation of fused proteins are known in the art. Several intracellular targeting sequences are described in Williams W V, Madaio M, Weiner D B. 2001 U.S. Pat. No. 6,248,565 B1 and are included herein by reference. Several antigen presentation pathway targeting molecules are described in Leifert J A, Rodriguez-Carreno M P, Rodriguez F, Whitton J L 2004 Immunological reviews 199: 40-53 and Lemieux, Supra 2002 and are included herein by reference. Some of these are summarized in Table 3.
For example, it has been demonstrated that targeting heterologous proteins to various intracellular destinations including, but not limited to, secreted (e.g. TPA; Zhongming L, Howard A, Kelley C, Delogu G, Collins F, Morris S. 1999 Infect Immun. 67: 4780-4786), membrane-anchored (e.g. human alkaline phosphatase (PLAP; Gerber L, Kodukula K, Udenfriend S. 1992 J Biol Chem 267: 12168-12173), endosome (e.g. human Lampl; Wu, T, Guarnieri F G, Staveley-O'Carroll K F, Viscidi R P, Levitsky H I, Hedrick L, Cho K R, August J T, Pardoll D M. 1995 Proc. Natl. Acad. Sci. 92: 11671-11675), proteosome (e.g. mouse Ubiquitin A76; Delogu G, Howard A, Collins F M, and Morris S L. 2000 Infect. Immun. 68: 3097-3102), or endoplasmic reticulum (Xu W, Chu Y, Zhang R, Xu H, Wang Y. Xiong S 2005 Virology 334: 255-263) alter or enhance immune responses. Endosomal targeting promotes a MHC class II response, while the destabilizing ubiquitin molecule (UbiquitinA76 versus native UbiquitinG76) is utilized to enhance entry into proteosomal degradation pathway and MHC class I presentation.
It is also well known in the art that the effects of intracellular targeting are antigen specific, and the optimal intracellular destination to create the desired immune response need to be determined empirically for each antigen.
Despite the improvements in immunogenicity obtained by fusion of an antigen to such targeting sequences, efficacy has not been obtained in humans or other primates using the modified antigens.
A further incremental improvement is to immunize with a mix of DNA Vaccine plasmids, each encoding different forms of antigen. Pavlakis G N, Gragerov A, and Felber B K. 2004 US Patent Application 2004/0241140 proposes that combinations of antigens applied at different sites and also at different times may increase protective immunity and suggest that using different forms of DNA sequentially or in combinations but applied at different sites may reproduce the improved immunogenicity obtained with other prime-boost vaccine combinations. They teach that combinations of vectors expressing different forms of antigens show improved immunogenicity especially when injected in different sites on the same mouse, compared to a mix of DNA vectors injected in the same site. The authors speculate that the efficiency of heterologous boosting of a DNA prime may be due to the different antigen presentation afforded by the heterologous vector or purified protein. The authors utilized secreted, cytoplasmic and proteosomal degraded versions of the antigen, and do not teach to use other targeting sequences, or to use a vector family or rationale strategy to facilitate determination of the optimal immunization cocktail.
Other investigators have also reported improved responses when two plasmids targeting an antigen to different cellular destinations were combined in immunization. Immunization with DNA vaccines using combinations of either secreted and cytoplasmic targeted antigen (Piechocki M P, Pilon S A, Wei W Z. 2001 J Immunol. 167:3367-3374), or cytoplasmic and ubiquitin conjugated forms of papillomavirus capsid genes (Liu W J, Zhao K N, Gao F G, Leggatt G R, Fernando G J P, Frazer I H. 2001 Vaccine 20: 862-869; Frazer I H. 2004 US Patent Application 2004/0241177]) or soluble or membrane anchored forms of HSV-2 glycoprotein D (Flo J 2003 Vaccine 21: 1239-1245) enhanced immune responses. These authors do not teach to use other targeting sequences, or to use a vector family to determine the optimal immunization cocktail.
Other investigators have reported no improvement in responses when two plasmids targeting an antigen to different cellular destinations were combined in immunization. For example, no enhancement of immune response was observed with a combination of cytoplasmic and endosomally targeted p55Gag (Marques E T A, Chikhlikar P, de Arruda L B, Leao I C, Lu Y, Wong J, Chen J S, Byrne B, August J T 2003 J Biol. Chem. 278: 37926-37936). However, when DNA vaccine plasmids targeting human immunodeficiency virus-1 gag to cytoplasmic or endosomal destinations were utilized in prime boost studies, endosome priming, cytoplasmic boosting gave immune responses that were similar or even stronger compared to endosome priming and boosting (and much stronger than cytoplasmic priming and boosting). The potential benefit of prime boost immunization with 2 plasmids with different targeting of the same antigen was not proposed or otherwise taught by the authors (Barros De Arruda L, Chickhlikar P R, August J T, and Marques E T A. 2004 Immunol. 112:126-33).
These studies teach that the optimal combination of plasmids for optimal immune protection will be antigen and delivery method specific, and will need to be determined for each antigen. The optimal presentation is also anticipated to be antigen and delivery specific. EP targets muscle cells and likely delivers antigens for immune presentation via cross presentation to APC. This may optimally require secreted and/or stable protein in the donor cell, along with cell death to attract and stimulate APC's. For Gene Gun, which targets dendritic cells, direct priming may be the dominant antigen presentation mode and proteosomal and/or endosomal targeted antigen, for enhanced MHCI or MHCII presentation, respectively, may be optimal. Combinations of plasmids, optimized for each modality, may ultimately provide superior protection. For example combinations of vectors expressing native, dendritic- and proteosomal-targeted SIV antigens provided superior protection in rhesus macaques (Rosati M, von Gegerfelt A, Roth P, ALicea C, Valentin A, Robert-Guroff M, Venzon D, Montefiori D C, Markham P, Felber B K, Pavlakis G N. 2005 J Virol. 79: 8480-8492); similar enhancement was observed with mixed plasmids encoding cytoplasmic and ubiquitin conjugated papillomavirus capsid genes (Liu et al. Supra, 2001). A rational method to determine the optimal presentation is not taught in the art.
As well, the art teaches that interpretation of existing mixed plasmid immunization results is uncertain, due the variations between vector backbone used in these examples. Targeting vectors, that allow fusion of a target antigen to different intracellular targeting sequences, such as those utilized above, or those described in Williams et al, Supra, 2001, and Bucht G, Sjolander K B, Eriksson S, Lindgren L, Lundkvist A, Elgh F. 2001 Vaccine 19: 3820-3829 are not optimally designed to determine immune responses to antigens targeted to various intracellular destinations. First, these vectors use standard typeII restriction enzyme cloning sites, for introduction of the antigen gene. The art teaches that small sequence variations between vector backbone can alter expression levels (Hartikka J, Sawdey M, Cornefert-Jensen F, Margalith M, Barnhart K, Nolasco M, Vahlsing H L, Meek J, Marquet M, Hobart P, Norman J, Manthorpe M. 1996 Hum Gene Ther. 7:1205-1217); different expression levels have been shown to influence the resulting immune response (Zinckgraf J W, Silbart L K 2003 Vaccine 21: 1640-1649). As well small peptide additions to recombinant proteins from cloning sites or peptide tags can alter the protein subcellular localization, even when these sequences do not contain targeting tags (Ramanathan M P, Ayyavoo V, Weiner D B. 2001 DNA Cell Biol 20: 101-105).
The targeting vectors of Williams et al, Supra, 2001 or Bucht et al, Supra, 2001, as well as other current DNA vaccine plasmids such as VR1012, also were constructed using standard typeII restriction enzyme cloning. This strategy, which utilizes nearest flanking useful restriction sites to move fragments into the vector ensures that the final vectors contain large amounts of extraneous sequences. These vectors do not comply with current WHO or FDA guidelines regarding content and elimination of extraneous materials. For example, VR1012 includes potentially detrimental sequences such as potentially recombinogenic transposon termini as an artifact of cloning the kanamycin resistance gene. Oligo-pyrimidine or oligo-purine sequences in a plasmid have been shown to increase dimer formation in a pUC plasmid, presumably through formation of unusual DNA structures such as a triple helix (Kato M. 1993 Mol Biol Rep. 18:183-187). VR1012 contains such sequences, as a polyG polyC tail used to join two fragments. Overall, a number of elements in bacterial derived sequences from prokaryotic plasmids have been shown to negatively affect gene expression in eukaryotic cells (Leite J P, Cousin C, Heysen A, D'Halluin J C. 1989 Gene. 82:351-356; Peterson D O, Beifuss K K, Morley K L. 1987 Molec Cell Biol 7:1563-1567) or bind eukaryotic transcription factors (Tully D B, Cidlowski J A. 1987 Biochem Biophys Res Commun. 144:1-10; Ghersa P, Whelan J, Pescini R, DeLamarter J F, Hooft van Huijsduijnen R. 1994 Gene 151:331-332; Kushner P J, Baxter J D, Duncan K G, Lopez G N, Schaufele F, Uht R M, Webb P, West B L. 1994 Mol. Endocrinol. 8:405-407), ultimately decreasing the performance of the vectors. The presence of chi sites in plasmids have been shown to promote dimerization (Zaman M M, Boles T C. 1996 J Bacteriol. 178:3840-3845). Plasmid nicking may be associated with AT rich regions that ‘breathe’ and are susceptible to endogenous single stranded nucleases. Palindrome sequences are unstable, as are direct or inverted repeats and Z DNA forming sequences that are deleted or rearranged by the E. coli host. Unusual secondary structure DNA includes runs of potentially Z DNA-forming alternating pyrimidine/purine sequences (such as CpG sequences; Bichara M, Schumacher S, and Fuchs R P. 1995 Genetics 140:897-907), G-rich sequences that may form tetraplex structures, and oligopyrimidine or oligopurine sequences that may form triplex DNA.
Elimination of extraneous DNA is essential to reduce the chance of inclusion of such spurious binding sites. However, when DNA is eliminated, other problems can arise. For example, prokaryotic replication tends to terminate in the Cytomegalovirus (CMV) promoter. Termination is enhanced when the orientation of the origin is close to, and parallel with the CMV promoter. Interference is perhaps due to secondary structure, or fortuitous binding of bacterial protein. New minimized DNA vaccine vectors such as pVAX1 contain the CMV promoter and replication origin in close proximity due to size reduction; this vector produces replication intermediates, thus reducing the quality of plasmid produced in the bacterial host (Levy J. 2003 US Patent Application US2003180949).
Class IIS restriction enzymes provide the means to digest DNA molecules outside of the restriction endonuclease recognition sites, making it possible to introduce a site (by PCR) and to digest it, creating an overhanging terminus with specific address tag at any point. By combining this characteristic with gene amplification technology, class IIS sites in the PCR primers can digest DNA at any site, providing unique, non-palindromic overhanging ends, or specific address tags (Lebedenko E N, Birikh K R, Plutalov O V, Berlin YuA. 1991 Nucleic Acids Res 19: 6757-61). The Gene Self-Assembly process uses class IIS restriction enzymes to generate unique, non-palindromic overhanging termini that can ligate to only one other terminus in a complex mixture, thus assuring that each fragment ligates in the correct orientation to its correct partner, and none other. Nature Technology Lincoln Nebr. has developed a Gene Self Assembly vector, pWizBang, that can be used to create unique, non-palindromic address labels on a series of DNA molecules (such as blunt restriction fragments, or PCR amplicons), permitting a number of fragments (up to 32) to be instantly ligated into a single, complex construct at once. This step permits modular vector construction, and eliminates sequential sub-cloning. An advantage of this technique is that it is seamless—fragments can be joined at any base without the need for restriction sites at these loci (Hodgson, C, Zink, M A, Xu, G., U.S. Pat. No. 6,410,220) eliminating all extraneous sequences. However, Class IIS vector development methodologies have not been applied to creation of optimized DNA vaccine plasmids.
Even in view of the prior art, there remains a need for improved vectors that are minimized to eliminate extraneous DNA, organized to ensure high quality bacterial plasmid productivity and improved in vivo expression, improved innate and adaptive immune response induction, and designed to facilitate rapid and rationale evaluation of mixed plasmid immunization, such that this technology can be utilized to meet the efficacy threshold in humans and other mammals, birds or fish with a wide range of target antigens.